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N J Maeji

Publications and source records attributed to N J Maeji.

13 recordsLinked to original sources
PubMed1995

Inhibitors of human heart chymase based on a peptide library.

We have synthesized two sets of noncleavable peptide-inhibitor libraries to map the S and S' subsites of human heart chymase. Human heart chymase is a chymotrypsin-like enzyme that converts angiotensin I to angiotensin II. The first library consists of peptides with 3-fluorobenzylpyruvamides in the P1 position. (Amino acid residues of substrates numbered P1, P2, etc., are toward the N-terminal direction, and P'1, P'2, etc., are toward the C-terminal direction from the scissile bond.) The P'1 and P'2 positions were varied to contain each one of the 20 naturally occurring amino acids and P'3 was kept constant as an arginine. The second library consists of peptides with phenylalanine keto-amides at P1, glycine in P'1, and benzyloxycarbonyl (Z)-isoleucine in P4. The P2 and P3 positions were varied to contain each of the naturally occurring amino acids, except for cysteine and methionine. The peptides of both libraries are attached to a solid support (pins). The peptides are evaluated by immersing the pins in a solution of the target enzyme and evaluating the amount of enzyme absorbed. The pins with the best inhibitors will absorb most enzyme. The libraries select the best and worst inhibitors within each group of peptides and provide an approximate ranking of the remaining peptides according to Ki. Through this library, we determined that Z-Ile-Glu-Pro-Phe-CO2Me and (F)-Phe-CO-Glu-Asp-ArgOMe should be the best inhibitors of chymase in this collection of peptide inhibitors. We synthesized the peptides and found Ki values were 1 nM and 1 microM, respectively. The corresponding Ki values for chymotrypsin were 10 nM and 100 microM. The use of libraries of inhibitors has advantages over the classical method of synthesis of potential inhibitors in solution: the libraries are reusable, the same libraries can be used with a variety of different serine proteases, and the method allows the screening of hundreds of compounds in short periods of time.

Amino Acid Sequence↗
PubMed1993

Systematic study of substance P analogs. I. Evaluation of peptides synthesized by the multipin method for quantitative receptor binding assay.

The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large-scale screening of oligopeptides [Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002]. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated. Substance P and 18 analogs, including a set of N-terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 +/- 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against 125I-BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50 values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high-volume screening of bioactive peptides.

Amino Acid Sequence↗
PubMed1993

Systematic study of substance P analogs. II. Rapid screening of 512 substance P stereoisomers for binding to NK1 receptor.

Recent developments in multiple peptide synthesis have made it feasible to synthesize and screen large numbers of peptide analogs simultaneously. We report here a model study of large scale screening of stereoisomers of substance P with systematic D-amino acid replacements. Such studies are useful in exploring conformational requirements of peptide-receptor interaction and to provide empirical information for peptide drug design. 512 stereoisomers of SP were prepared by the multipin peptide synthesis method. Receptor binding affinities of these analogs were estimated by an iterative competition assay. Results obtained form a comprehensive database on the stereochemical requirement of SP binding to central NK1 receptor. Data from analogs with single D-amino acid replacement are consistent with those previously reported showing that SP binding is highly sensitive to the chirality change of the C-terminal residues (Gln6-Leu10), but less sensitive to the chirality change of the N-terminal residues (Arg1-Gln5). A qualitative analysis of the database by comparison of series of peptide pairs revealed a repeated pattern of affinity change with D-amino acid replacement, suggesting a largely additive binding activity of SP from each residue. On the other hand, possible intramolecular interactions between some N-terminal and C-terminal residues to form an optimal binding conformation were also found. A set of 189 peptides with IC50 values less than 5 microM was subjected to an empirical QSAR analysis using a linear additive model. The relative contribution coefficients obtained agreed with the observation that the predominant contribution comes from the C-terminal residues, suggesting considerable independency of each residue on binding to NK1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Simultaneous multiple synthesis of peptide-carrier conjugates.

Recently, the multipin approach for simultaneous multiple peptide synthesis was applied to the analysis of T cell determinants by using a novel cleavage method (Maeji et al., 1990). A diketopiperazine forming linker allowed cleavage of peptides into aqueous buffer which, without further purification, could be used immediately in cell culture assays. Another potential application of the technique is the simultaneous cleavage and coupling of peptides to immunogenic carriers. Without further purification the resulting conjugates can be used for the production of antipeptide antisera. The choice of carrier and conjugation chemistry is not restricted as peptide/pin cleavage occurs in aqueous solution over a range of pH and ionic strength. The method was assessed using the 2,4-dinitrophenyl group as a model hapten, diphtheria toxoid as the carrier, and N-(epsilon-maleimidocaproyloxy)succinimide as the cross-linking reagent. The resulting DNP-DT conjugate was used to prepare high titered specific anti-DNP antisera in mice.

2,4-Dinitrophenol↗
PubMed1991

Synthesis of peptide analogues using the multipin peptide synthesis method.

Modification of the multipin peptide synthesis method which allows the simultaneous synthesis of large numbers of different peptide analogues is described. Peptides were assembled on polyethylene pins derivatized with a 4-(beta-alanyloxymethyl)benzoate (beta-Ala-HMB) handle. For comparative purposes, peptides were also assembled on the diketopiperazine-forming handle N epsilon-(beta-alanyl)lysylprolyloxylactate. In model studies it was demonstrated that beta-Ala-HMB-linked peptides were cleaved from polyethylene pins with dilute sodium hydroxide or 4% methylamine/water to yield analogues with beta-Ala-free acid (beta-Ala-CO2H) and beta-Ala-methylamide (beta-Ala-CONHCH3), respectively. To assess the suitability of this approach for T-cell determinant analysis, analogues of a known T-cell determinant were synthesized with the various C-terminal endings. Peptides were characterized by amino acid analysis and fast atom bombardment-mass spectrometry. HPLC of the crude cleaved peptides indicated that 22 of the 24 peptides were greater than 95% pure. These crude peptide solutions were nontoxic in sensitive cell culture assays without further purification. All three cleavage procedures gave comparable activities in T-cell proliferation assays. These results demonstrate the potential of the multipin peptide synthesis method for the production of large numbers of different peptide analogues.

Amino Acid Sequence↗
PubMed1990

Multi-pin peptide synthesis strategy for T cell determinant analysis.

Techniques to synthesize many peptides simultaneously exist, however their individual cleavage and subsequent purification constitutes a bottleneck to total throughput. Biological screening of peptides is generally carried out at physiological pH in aqueous solutions. However, peptides, unless individually purified are usually contaminated by residual compounds used in their preparation such as trifluoroacetic acid, organic solvents, scavengers etc. In testing with cellular systems, such as T cell determinant analysis, such contaminations must be rigorously excluded. We have extended the pin synthesis technique of synthesizing and screening large number of peptides (Geysen et al., 1984) to the analysis of T cell determinants. Peptides can be synthesized on polyethylene pins, the side chain protective groups removed and the peptides washed free of contaminants. A linker system stable under these conditions can then be triggered to cleave the peptides from the pins in an aqueous solution at neutral pH. This strategy enables the rapid mapping of T cell determinants. It is also applicable to other systems where large numbers of solution phase peptides are required, for example, in the study of hormone analogues.

Amino Acid Sequence↗
PubMed1989

N.m.r. study on conformation of Ac-Thr(alpha-GalNAc)-Ala-Ala-OMe as a model for mucin type glycoprotein.

This study report on the results of high resolution 1H n.m.r. investigations on Ac-Thr(alpha-GalNAc)-Ala-Ala-OMe 1 as a mucin type model glycopeptide of antifreeze glycoprotein (AFGP) in both dimethyl sulfoxide (DMSO) and H2O. The temperature dependence of amide proton chemical shifts strongly suggested the presence of the intramolecular hydrogen bond between the amide proton of GalNAc and the carbonyl oxygen of the Thr residues. Due to this bond, the orientation of the sugar residue of 1 appears to be fairly restricted relative to its peptide backbone. Despite the lack of the clear evidence for such intramolecular hydrogen bond in H2O, 1H coupling constant data suggested the structural similarity of 1 in DMSO and H2O, indicating the presence of the intramolecular hydrogen bond even in H2O, which may play an important role in determining the orientation of the sugar moiety with respect to the peptide backbone in glycoprotein.

Amino Acid Sequence↗
PubMed1987

Conformational study of O-glycosylated threonine containing peptide models.

1H n.m.r. studies of Z-Thr-OMe, Z-Thr-Ala-OMe, Z-Ala-Thr-OMe and their glycosylated derivatives indicate the possibility of an intramolecular hydrogen bond between Thr N alpha H and the N-acetyl carbonyl of the carbohydrate moiety, 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-galactopyranose (AcGalNAc). This is especially true in the case of Z-Thr(AcGalNAc)-Ala-OMe, suggesting that the strength of this hydrogen bond is dependent on the neighboring amino acids on the carbonyl terminal side of Thr. The existence of such a hydrogen bond implies a conformation in which the carbohydrate moiety is restricted to an orientation with its plane roughly perpendicular to the peptide backbone. In such an orientation, steric problems will be minimized in the case of clustered O-glycosidically linked Thr(Ser) residues as found in human erythrocyte glycophorin. A locked orientation of the carbohydrate moiety with respect to the peptide backbone may also play a conformational role in antifreeze glycoproteins.

Glycopeptides↗
PubMedDate not supplied

Use of the multipin peptide synthesis technique for the generation of antipeptide sera.

The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes. What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants. A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies. This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pin-synthesized peptides and beta-amide peptides and the protein antigen coated to microtiter plates.

Amino Acid Sequence↗
PubMedDate not supplied

Systematic screening for bioactive peptides.

Using simultaneous multiple peptide synthesis by the multipin approach, the feasibility of systematic large-scale pharmacological screening of peptide ligands was investigated. The method involves the assembly of small quantities of peptides (ca. 50 nmol) on plastic pins derivatized with an ester linker based on glycolate and 4-(hydroxymethyl)benzoate. These esters are stable under peptide synthesis and side-chain deprotection conditions but cleave under relatively mild basic conditions to generate peptides having C-terminal acid, amide and methylamide. A two-step approach to side-chain deprotection and cleavage from the solid support allows potentially toxic reagents to be removed (washed) from the peptide prior to cleavage. Consequently, the resulting peptide solutions can be used in bioassays with minimal processing. A series of angiotensin II and substance P analogs were synthesized and evaluated in an in vivo rat model and in vitro radioreceptor assay, respectively. Structure-activity studies on analogs of these bioactive peptides are well documented. The data obtained were consistent with that reported in the literature.

Amino Acid Sequence↗
PubMedDate not supplied

Multipin technology in the preparation and screening of peptide libraries.

Peptide libraries are relatively new sources of enormous numbers of unique compounds, fodder for the mill of drug discovery programs. Their enormous diversity derives from vast numbers of combinations of a small number of monomers (Geysen et al., 1986). For example, a complete hexapeptide library synthesized from just 10 monomers (amino acids) has one million unique compounds in it. In principle, other types of combinatorial libraries can have equally vast numbers of members; for example, the monomers can be N-acyl glycines, giving rise to the so-called "peptoids" (Simon et al., 1992); or the monomers could be monosaccharides or nucleotides.

Biotechnology↗
PubMedDate not supplied

Larger scale multipin peptide synthesis.

The multipin peptide synthesis approach originated as an immunological tool for epitope mapping. However, continuing evolution of the basic technology has allowed synthesis at scales up to 10 mumol per pin. At this loading, the methodology can no longer be considered just a screening tool. The overall synthesis efficiency of this approach was assessed by the synthesis of 2913 different peptides having little or no sequence homology and ranging up to a 46-mer in length. High performance liquid chromatography analysis of the crude peptides indicates overall quality of synthesis was high. The method is suitable for multi-milligram synthesis of peptides without sacrificing any of the inherent advantages of the 96-well format.

Amino Acid Sequence↗